1. Field of the Invention
This invention relates to the identification of polypeptides which have been separated on the same gel, typically from polyacrylamide gel electrophoresis (PAGE) and to a kit for use in the method. It is especially useful in proteomics (the large scale identification and characterization of proteins).
2. Description of the Related Art
In proteomics, massively parallel protein identification and characterization techniques are required. The identification of proteins or other polypeptides merely by PAGE, even using two-dimensional gels (2D-PAGE), is laborious and often uncertain. Many different methods have been developed to identify and partially characterize proteins from complex biological samples. Some of them use Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) techniques to analyze peptide xe2x80x9cfingerprintsxe2x80x9d produced by fragmenting the proteins with enzymes. Several software programs have been developed to compare mass spectra of the peptides obtained from MALDI-TOF experiments with theoretical spectra from proteins. The subject has been reviewed by Kussmann and Roepstorff [1]. These authors noted three ways in which proteins separated by gel electrophoresis could be digested with enzymes to yield fragment peptides:
1. The digestion can be carried out in a plug of excised gel and the peptides recovered by elution. This is the authors"" own preference.
2. The protein can be first electroleluted from an excised gel plug and then digested in solution.
3. The protein can be electroblotted onto a membrane and subsequently digested on the membrane.
These types of processes are not practical for the sequencing of polypeptides which have been run on the same gel, since the cutting out of the polypeptide bands from the gel has to be done sequentially and the plugs thus obtained placed in tubes for further analysis. Also, losses occur when the polypeptides adhere to the walls of the tube.
The present invention provides a solution to the above problem. It has now been found that the proteins or other polypeptides separated on a gel can be cleaved into fragments, for example by digestion with an enzyme, and these fragments presented very satisfactorily for analysis, especially by MALDI-TOF MS, if the cleaving agent is immobilized and interposed as the xe2x80x9cfillingxe2x80x9d in an electroblotting xe2x80x9csandwichxe2x80x9d between the separation gel as one xe2x80x9cslicexe2x80x9d of the sandwich and a collection layer, exemplified as a conventional PVDF membrane, as the other xe2x80x9cslicexe2x80x9d of the sandwich. In this way, the fragments are collected on the hydrophobic layer and can then be formulated in an appropriate way for the MALDI-TOF MS. It is only necessary that the electroblotting is carried out so that the proteins have a long enough residence period in the proximity of the immobilized cleaving agent to ensure that a reasonable amount of the fragments is produced, but, of course, not so long as to allow undesired diffusion. This is easily achievable by varying appropriately the current used in the electroblotting, e.g. by pulsing the current or using a unsymmetrical alternating current. Further, when an enzyme is used as the cleaving agent and when the enzyme is immobilized securely on the hydrophilic membrane, especially by covalent bonding to the solid phase, autodigestion (cleavage of the enzyme by itself) is inhibited.
According to a specific aspect of the invention there is provided a method of identifying polypeptides which have been separated on the same gel by electrophoresis, comprising the steps of:
a) providing adjacent to the gel at least one hydrophilic membrane on which is immobilized at least one reagent capable of cleaving a polypeptide;
b) providing a hydrophobic collection layer suitable for receiving thereon fragments of polypeptide transferred thereto by electroblotting, said hydrophobic layer being positioned beyond the hydrophilic membrane in a direction of movement of the fragments of polypeptide (usually cathode to anode);
c) electroblotting the polypeptides from the separation gel through the hydrophilic membrane or membranes, under conditions effective to cause the polypeptides to be cleaved into fragments by the cleaving reagent, to the hydrophobic layer; and
d) identifying the fragments collected on the hydrophobic layer and from the identification of the fragments, identifying the polypeptide from which they came.
In another aspect, the invention provides a membrane suitable for use in an electroblotting sandwich, having immobilized thereon at least one polypeptide-cleaving reagent, especially a modified PVDF membrane having a protease, especially trypsin, covalently bonded thereto.
The invention further includes a kit for use in the method of the invention, said kit comprising:
a) at least one hydrophilic membrane on which is immobilized at least one reagent capable of cleaving a polypeptide; and
b) a hydrophobic collection layer suitable for receiving thereon fragments of polypeptide transferred thereto by electroblotting.
The term xe2x80x9ckitxe2x80x9d as used herein includes combinations of the identified components in separate containers and also an assembly of the hydrophilic membrane(s) and hydrophobic collection layer ready for use. The kit may further include reagents useful in the method of the invention, e.g. electroblotting buffer, reagent(s) which assist in the reaction of the enzyme with the polypeptide fragment and so on.
The term xe2x80x9ccollection layerxe2x80x9d as used herein has a broad meaning, since this is not in itself critical to the invention. It may be in isolation self-supporting or non-self-supporting and can be a membrane, film, coating or plate. It will normally be porous to the electroblotting buffer, to enable current to be carried to or from the electrode, but may alternatively be the electrode or in direct electrical communication with it.
The term xe2x80x9cidentifyingxe2x80x9d as used herein is not synonymous with determining the sequence and includes partially identifying the polypeptide or characterizing it as similar to or different from a known protein. Further it includes making a tentative identification based on the most probable of a small number of possibilities.